Cancer is a generic name for a wide range of cellular dysfunctions and dysregulations characterized by unregulated growth, lack of differentiation, and the potential to invade local tissues and metastasize to distant sites. These neoplastic malignancies may affect, with various degrees of prevalence, every tissue and organ in the body. The present invention is directed to methods for diagnosing and treating cancer patients. In particular, the present invention is directed to methods for determining which patients will most benefit from treatment with anti-cancer agents that are inhibitors of protein kinases, e.g. epidermal growth factor receptor (EGFR) kinase inhibitors (e.g. erlotinib), or IGF-1R kinase inhibitors (e.g. OSI-906), and methods of identifying and characterising new anti-cancer agents.
It has been recognized that inhibitors of protein kinases are useful as selective inhibitors of the growth of mammalian cancer cells. For example, Gleevec™ (also known as imatinib mesylate), a 2-phenylpyrimidine tyrosine kinase inhibitor that inhibits the kinase activity of the BCR-ABL fusion gene product, has been approved by the U.S. Food and Drug Administration for the treatment of CML. The 4-anilinoquinazoline compound Tarceva™ (erlotinib HCl) has also been approved by the FDA, and selectively inhibits EGF receptor kinase with high potency. The development for use as anti-tumor agents of compounds that directly inhibit the kinase activity of IGF-1R, as well as antibodies that reduce IGF-1R kinase activity by blocking IGF-1R activation or antisense oligonucleotides that block IGF-1R expression, are areas of intense research effort (e.g. see Larsson, O. et al (2005) Brit. J. Cancer 92:2097-2101; Ibrahim, Y. H. and Yee, D. (2005) Clin. Cancer Res. 11:944s-950s; Mitsiades, C. S. et al. (2004) Cancer Cell 5:221-230; Camirand, A. et al. (2005) Breast Cancer Research 7:R570-R579 (DOI 10.1186/bcr1028); Camirand, A. and Pollak, M. (2004) Brit. J. Cancer 90:1825-1829; Garcia-Echeverria, C. et al. (2004) Cancer Cell 5:231-239; Sachdev D, and Yee D., Mol Cancer Ther. 2007 January; 6(1):1-12; Hofmann F., and Garcia-Echeverria C., Drug Discov Today 2005 10:1041-7). Agents inhibiting the IGF-1R pathway have demonstrated anti-tumor efficacy in multiple human cancer models both in vitro and in vivo, particularly in pediatric models of Ewing's sarcoma and rhabdomyosarcoma (Manara M C, et al. Int J Oncol 2005 27:1605-16). Despite early hints of efficacy in patients with sarcoma, results to date of IGF-1R inhibitors in early clinical trials have not been impressive, indicating that patient selection strategies and rational combinations may be needed to move forward with this approach (Tolcher A. W., et al. Journal of Clinical Oncology, 2007 ASCO Annual Meeting Proceedings Part I. Vol 25, No. 18S (June 20 Supplement), 2007: 3002). Data acquired this far, has not indicated that activation, overexpression, or amplification of members of the IGF-1R pathway will predict responsiveness.
The epidermal growth factor receptor (EGFR) family comprises four closely related receptors (HER1/EGFR, HER2, HER3 and HER4) involved in cellular responses such as differentiation and proliferation. Over-expression of the EGFR kinase, or its ligand transforming growth factor-alpha (TGF-alpha), is frequently associated with many cancers, including breast, lung, colorectal, ovarian, renal cell, bladder, head and neck cancers, glioblastomas, and astrocytomas, and is believed to contribute to the malignant growth of these tumors. A specific deletion-mutation in the EGFR gene (EGFRvIII) has also been found to increase cellular tumorigenicity. Activation of EGFR stimulated signaling pathways promote multiple processes that are potentially cancer-promoting, e.g. proliferation, angiogenesis, cell motility and invasion, decreased apoptosis (programmed cell death) and induction of drug resistance. Increased HER1/EGFR expression is frequently linked to advanced disease, metastases and poor prognosis. For example, in non small cell lung cancer (NSCLC) and gastric cancer, increased HER1/EGFR expression has been shown to correlate with a high metastatic rate, poor tumor differentiation and increased tumor proliferation.
Mutations which activate the EGF receptor's intrinsic protein tyrosine kinase activity and/or increase downstream signaling have been observed in NSCLC and glioblastoma. However the role of mutations as a principle mechanism in conferring sensitivity to EGFR kinase inhibitors, for example erlotinib (TARCEVA®) or gefitinib (IRESSA™), has been controversial. Recently, a mutant form of the full length EGFR has been reported to predict responsiveness to the EGFR tyrosine kinase inhibitor gefitinib (Paez, J. G. et al. (2004) Science 304:1497-1500; Lynch, T. J. et al. (2004) N. Engl. J. Med. 350:2129-2139). Cell culture studies have shown that cell lines which express the mutant form of EGFR (i.e. H3255) were more sensitive to growth inhibition by the EGFR tyrosine kinase inhibitor gefitinib, and that much higher concentrations of gefitinib was required to inhibit the tumor cell lines expressing wild type EGFR. These observations suggests that specific mutant forms of EGFR may reflect a greater sensitivity to EGFR kinase inhibitors, but do not identify a completely non-responsive phenotype.
The development for use as anti-tumor agents of compounds that directly inhibit the kinase activity of EGFR, as well as antibodies that reduce EGFR kinase activity by blocking EGFR activation, are areas of intense research effort (de Bono J. S. and Rowinsky, E. K. (2002) Trends in Mol. Medicine. 8:S19-S26; Dancey, J. and Sausville, E. A. (2003) Nature Rev. Drug Discovery 2:92-313). Erlotinib (e.g. erlotinib HCl, also known as TARCEVA® or OSI-774) is an orally available inhibitor of EGFR kinase. In vitro, erlotinib has demonstrated substantial inhibitory activity against EGFR kinase in a number of human tumor cell lines, including colorectal and breast cancer (Moyer J. D. et al. (1997) Cancer Res. 57:4838), and preclinical evaluation has demonstrated activity against a number of EGFR-expressing human tumor xenografts (Pollack, V. A. et al (1999) J. Pharmacol. Exp. Ther. 291:739). More recently, erlotinib has demonstrated promising activity in Phase I and II trials in a number of indications, including head and neck cancer (Soulieres, D., et al. (2004) J. Clin. Oncol. 22:77), NSCLC (Perez-Soler R, et al. (2001) Proc. Am. Soc. Clin. Oncol. 20:310a, abstract 1235), colorectal cancer (CRC) (Oza, M., et al. (2003) Proc. Am. Soc. Clin. Oncol. 22:196a, abstract 785) and metastatic breast cancer (MBC) (Winer, E., et al. (2002) Breast Cancer Res. Treat. 76:5115a, abstract 445). In a Phase III trial, erlotinib monotherapy significantly prolonged survival, delayed disease progression and delayed worsening of lung cancer-related symptoms in patients with advanced, treatment-refractory NSCLC (Shepherd, F. et al. (2004) J. Clin. Oncology, 22:14 S (July 15 Supplement), Abstract 7022). While most of the clinical trial data for erlotinib relate to its use in NSCLC, preliminary results from Phase I/II studies have demonstrated promising activity for erlotinib and capecitabine/erlotinib combination therapy in patients with wide range of human solid tumor types, including CRC (Oza, M., et al. (2003) Proc. Am. Soc. Clin. Oncol. 22:196a, abstract 785) and MBC (Jones, R. J., et al. (2003) Proc. Am. Soc. Clin. Oncol. 22:45a, abstract 180). In November 2004 the U.S. Food and Drug Administration (FDA) approved TARCEVA® for the treatment of patients with locally advanced or metastatic NSCLC after failure of at least one prior chemotherapy regimen. TARCEVA® is the only drug in EGFR class to demonstrate in a Phase III clinical trial an increase in survival in advanced NSCLC patients.
IGF-1R belongs to the insulin receptor family that includes the Insulin Receptor (IR), IGF-1R (homodimer), IGF-1R/IR (hybrid receptor), and IGF-2R (mannose 6-phosphate receptor). IGF-1R/IR hybrids act as homodimers, preferentially binding and signaling with IGFs. IR exists in two isoforms: IR-B (traditional insulin receptor) and IR-A (a fetal form which is re-expressed in selected tumors and preferentially binds IGF-II). IGF-2R is a non-signaling receptor that acts as a “sink” for IGF-II (Pollak M. N., et al. Nat Rev Cancer 2004 4:505-18). Six well-characterized insulin-like growth factor binding proteins (IGFBP-1 through -6) associate with IGF ligands to stabilize the IGFs and modulate their ability to bind the IGF-1R.
IGF-1R is a transmembrane RTK that binds primarily to IGF-1 but also to IGF-II and insulin with lower affinity. Binding of IGF-1 to its receptor results in activation of it's tyrosine kinase activity, intermolecular receptor autophosphorylation, and phosphorylation of cellular substrates, including IRS1 and Shc, leading to activation of the PI3K/Akt and mitogen-activated protein kinase (MAPK) pathways (Adams T. E., et al. Cell Mol Life Sci 2000 57:1050-93; Pollak M. N., et al. Nat Rev Cancer 2004 4:505-18; Baserga R., Exp Cell Res 1999 253:1-6). The ligand-activated IGF-1R induces mitogenic activity in normal cells and plays an important role in abnormal growth. A major physiological role of the IGF-1 system is the promotion of normal growth and regeneration. Overexpressed IGF-1R (type 1 insulin-like growth factor receptor) can initiate mitogenesis and promote ligand-dependent neoplastic transformation. Furthermore, IGF-1R plays an important role in the establishment and maintenance of the malignant phenotype. Unlike the epidermal growth factor (EGF) receptor, no mutant oncogenic forms of the IGF-1R have been identified. However, several oncogenes have been demonstrated to affect IGF-1 and IGF-1R expression. A correlation between a reduction of IGF-1R expression and resistance to transformation has been seen. Exposure of cells to mRNA antisense to IGF-1R RNA prevents soft agar growth of several human tumor cell lines. IGF-1R abrogates progression into apoptosis, both in vivo and in vitro. It has also been shown that a decrease in the level of IGF-1R below wild-type levels causes apoptosis of tumor cells in vivo. The ability of IGF-1R disruption to cause apoptosis appears to be diminished in normal, non-tumorigenic cells.
The IGF-1 pathway has an important role in human tumor development. IGF-1R overexpression is frequently found in various tumors (breast, colon, lung, sarcoma) and is often associated with an aggressive phenotype. High circulating IGF1 concentrations are strongly correlated with prostate, lung and breast cancer risk. Furthermore, IGF-1R is required for establishment and maintenance of the transformed phenotype in vitro and in vivo (Baserga R. Exp. Cell. Res., 1999, 253, 1-6). The kinase activity of IGF-1R is essential for the transforming activity of several oncogenes: EGFR, PDGFR, SV40 T antigen, activated Ras, Raf, and v-Src. The expression of IGF-1R in normal fibroblasts induces neoplastic phenotypes, which can then form tumors in vivo. IGF-1R expression plays an important role in anchorage-independent growth. IGF-1R has also been shown to protect cells from chemotherapy-, radiation-, and cytokine-induced apoptosis. Conversely, inhibition of endogenous IGF-1R by dominant negative IGF-1R, triple helix formation or antisense expression vector has been shown to repress transforming activity in vitro and tumor growth in animal models. The IGF-1R signaling pathway also appears to be a robust target in colorectal cancer (CRC), based upon data demonstrating overexpress ion of the receptor and ligands in CRC, association with a more malignant phenotype, chemotherapy resistance, and correlation with a poor prognosis (Saltz, L. B., et al. J Clin Oncol 2007; 25(30): 4793-4799; Tripkovic I., et al. Med. Res. 2007 July; 38(5):519-25. Epub 2007 Apr. 26; Miyamoto S., et al. Clin Cancer Res. 2005 May 1; 11(9):3494-502; Nakamura M., et al. Clin Cancer Res. 2004 Dec. 15; 10(24):8434-41; Grothey A, et al. J Cancer Res Clin Oncol. 1999; 125(3-4):166-73).
There is a need for both more efficacious treatment for neoplasia and other proliferative disorders, and for more effective means for determining which tumors will respond to which treatment. Several groups have investigated or disclosed potential biomarkers to predict a patient's response to protein-tyrosine kinase inhibitors (see for example, PCT publications: WO 2004/063709, WO 2005/017493, WO 2004/111273, WO 2008/108986, WO 2007/001868, WO 2004/071572, WO 2003/078662, WO 2007/067500, WO 2005/070020, WO 2009/015233, WO 2009/023172, WO 2004/046386, WO 2008/070460, and WO 2010/022268; US published patent applications: US 2005/0019785, US 2007/0065858, US 2005/0164218, US 2009/0092596, US 2009/0093488, US 2009/0093488, US 2006/0140960, US 2009/0118175, US 2004/0132097, US 2003/0165954, US 2007/0218512, US 2007/0265185, US 2007/0270505, US 2007/0128636, US 2009/0092596, US 2007/0212738, US 2007/0237770, US 2009/0029354, US 2009/0092526, US 2006/0263775, US 2004/0018528, US 2006/0121539, US 2008/0131885, US 2005/0019785, US 2006/0263806, US 2007/0172857, US 2004/0048254, US 2009/0061454, US 2009/0123374, US 2007/0196352, US 2006/0078941, US 2008/0234138, US 2005/0170386, US 2002/0169562, US 2003/0053995, US 2007/0077577, US 2008/0187930, US 2006/0003365, US 2005/0260664, US 2008/0112888, US 2008/0019961, US 2008/0167532, US 2006/0234259, US 2004/0063120, US 2007/0092881, US 2008/0026481, US 2009/0092983, US 2004/0214203, US 2009/0136945, US 2007/0154915, US 2009/0155786, US 2008/0015160, US 2008/0312093, US 2008/0176229, US 2004/0157255, US 2007/0031871, US 2009/0061422, US 2008/0113874, US 2006/0019268, US 2007/0065858, US 2007/0231822, and US 2009/0023149, and U.S. Pat. Nos. 5,367,064, 7,368,551, 6,171,779, 7,342,108, 6,413,730, 7,526,387, 6,271,363, 6,251,628, and 7,569,349). Several biomarkers have been proposed for predicting the response to EGFR kinase inhibitors, including mutant KRAS as a predictor of non-responsiveness in colorectal cancer (e.g. see Brugger, W. et al. (2009) J Clin Oncol 27:15s, (suppl; abstr 8020); Siena, S et al (2009) JNCI 101(19):1308-1324; Riely and Ladanyi (2008) J Mol Diagnostics 10(6):493; Jimeno, A. et al. (2009) Cancer J. 15(2):110-13). In addition, several biomarkers, including mutant KRAS, have been disclosed that have potential in predicting a patient's response to IGF-1R kinase inhibitors (e.g. see Rodon, J. et al (2008) Mol Cancer Ther. 7:2575-2588; T. Pitts et al. (2009) EORTC Conference, Boston, Mass., abstract #2141; Huang, F. et al. (2009) Cancer Res. 69(1):161-170; Rodon, J. et al., (2008) Mol. Cancer. Ther. 7:2575-2588). However, in most instances no FDA-approved diagnostic tests have yet emerged that can effectively guide practicing physicians in the treatment of their patients with such inhibitors, or can indicate to the physician which tumors will respond most favorably to a combination of such an inhibitor with a standard chemotherapy agent.
During most cancer metastases, an important change occurs in a tumor cell known as the epithelial-mesenchymal transition (EMT) (Thiery, J.P. (2002) Nat. Rev. Cancer 2:442-454; Savagner, P. (2001) Bioessays 23:912-923; Kang Y. and Massague, J. (2004) Cell 118:277-279; Julien-Grille, S., et al. Cancer Research 63:2172-2178; Bates, R. C. et al. (2003) Current Biology 13:1721-1727; Lu Z., et al. (2003) Cancer Cell. 4(6):499-515)). EMT does not normally occur in healthy cells except during embryogenesis, though a transient EMT state is induced in epithelial wound healing to aid in the reconstruction of epithelial tissue. Epithelial cells, which are bound together tightly and exhibit polarity, change to a more mesenchymal cellular phenotype, in which these mesenchymal cells are held together more loosely, exhibit a loss of polarity, and have the ability to move within tissues. These mesenchymal-like cells can spread into tissues surrounding the original tumor, as well as separate from the tumor, invade blood and lymph vessels, and travel to new locations where they divide and form additional tumors. Recent research has demonstrated that epithelial cells respond well to EGFR and insulin-like growth factor-1 receptor (IGF-1R) kinase inhibitors, but that after an EMT the resulting mesenchymal-like tumor cells are much less sensitive to such inhibitors. (e.g. see Thompson, S. et al. (2005) Cancer Res. 65(20):9455-9462; U.S. Patent Application 60/997,514). Thus there is a pressing need for anti-cancer agents that can prevent or reverse tumor cell EMT events (e.g. stimulate a mesenchymal to epithelial transition (MET)), or inhibit the growth of the mesenchymal-like tumor cells resulting from EMT. Such agents should be particularly useful when used in conjunction with other anti-cancer drugs such as EGFR and IGF-1R kinase inhibitors. The present invention provides new methods for identification and characterization of compounds that modulate EMT.
As human cancers progress to a more invasive, metastatic state, multiple signaling programs regulating cell survival and migration are observed depending on cell and tissue contexts (Gupta, G. P., and Massague, J. (2006) Cell 127, 679-695). Recent data highlight the transdifferentiation of epithelial cancer cells to a more mesenchymal-like state, a process resembling epithelial-mesenchymal transition (EMT; (Oft, M., et al. (1996). Genes & development 10, 2462-2477; Perl, A. K., et al. (1998). Nature 392, 190-193), to facilitate cell invasion and metastasis (Brabletz, T. et al. (2005) Nat Rev Cancer 5, 744-749; Christofori, G. (2006) Nature 441, 444-450). Through EMT-like transitions mesenchymal-like tumor cells are thought to gain migratory capacity at the expense of proliferative potential. A mesenchymal-epithelial transition (MET) has been postulated to regenerate a more proliferative state and allow macrometastases resembling the primary tumor to form at distant sites (Thiery, J. P. (2002) Nat Rev Cancer 2, 442-454). EMT-like transitions in tumor cells result from transcriptional reprogramming over considerable periods of time (weeks to months) via transcription factors harboring zinc finger, forkhead, bHLH and HMG-box domains (Mani, S. A. et al. (2007) Proceedings of the National Academy of Sciences of the United States of America 104, 10069-10074; Peinado, H. et al. (2007) Nat Rev Cancer 7, 415-428). The loss of E-cadherin and transition to a more mesenchymal-like state, with increased expression of mesenchymal proteins such as vimentin or fibronectin, likely serves a major role in the progression of cancer (Matsumura, T. et al. (2001) Clin Cancer Res 7, 594-599; Yoshiura, K. et al. (1995). Proceedings of the National Academy of Sciences of the United States of America 92, 7416-7419) and the acquisition of a mesenchymal phenotype has been correlated with poor prognosis (Baumgart, E. et al. (2007) Clin Cancer Res 13, 1685-1694; Kokkinos, M. I. Et al. (2007) Cells, tissues, organs 185, 191-203; Willipinski-Stapelfeldt, B. et al. (2005) Clin Cancer Res 11, 8006-8014.). Targeting tumor-derived and/or tumor-associated stromal cells provides a unique mechanism to block EMT-like transitions and inhibit the survival of invading cells.
The cellular changes associated with EMT-like transitions alter the dependence of carcinoma cells on EGFR signaling networks for survival. It has been observed that an EMT-like transition was associated with cellular insensitivity to the EGFR kinase inhibitor erlotinib (Thomson, S. et al. (2005) Cancer Research 65, 9455-9462; Witta, S. E., et al. (2006) Cancer Research 66, 944-950; Yauch, R. L., et al. (2005) Clin Cancer Res 11, 8686-8698), in part from EGFR independent activation of either or both the PI3-kinase or Mek-Erk pathways (Buck, E. et al. (2007). Molecular Cancer Therapeutics 6, 532-541). Similar data correlating EMT status to sensitivity to EGFR kinase inhibitors have been reported in pancreatic, CRC (Buck, E. et al. (2007) Molecular Cancer Therapeutics 6, 532-541) bladder (Shrader, M. et al. (2007) Molecular Cancer Therapeutics 6, 277-285) and HNSCC (Frederick et al. (2007) Molecular Cancer Therapeutics 6, 1683-1691) cell lines, xenografts and in patients (Yauch, R. L., et al. (2005) Clin Cancer Res 11, 8686-8698). The molecular determinants to alternative routes of activation of the PI3-kinase and Erk pathways, which can bypass cellular sensitivity to EGFR kinase inhibitors, have been actively investigated (Chakravarti, A. et al. (2002) Cancer research 62, 200-207; Engelman, J. A. et al. (2007) Science 316:1039-1043).
Although considerable progress has been made in recent years in elucidating factors that influence tumor cell sensitivity to EGFR or IGF-1R kinase inhibitors, there remains a critical need for improved methods for determining the best mode of treatment for any given cancer patient and for the incorporation of such determinations into more effective treatment regimens for cancer patients, whether such inhibitors are used as single agents or combined with other anti-cancer agents. The present invention provides new methods for determining which tumors will respond most effectively to treatment with such inhibitors.